Method of inducing immunity against stratum corneum chymotrytic enzyme

ABSTRACT

The disclosed nucleic acid primer sets, used in combination with quantitative amplification (PCR) of tissue cDNA, can indicate the presence of specific proteases in a tissue sample. The detected proteases are themselves specifically overexpressed in certain cancers, and their presence may serve for early detection of associated ovarian and other malignancies, and for the design of interactive therapies for cancer treatment.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application which claims the benefit of priority ofU.S. Ser. No. 09/502,600, filed Feb. 11, 2000, now U.S. Pat. No.6,294,344, which is a continuation-in-part application of U.S. Ser. No.09/039,211, filed Mar. 14, 1998, now U.S. Pat. No. 6,303,318.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Generally, the present invention relates to the fields of molecularbiology and medicine. More specifically, the present invention is in thefield of cancer, especially ovarian cancer diagnosis.

2. Background of the Invention

To date, ovarian cancer remains the number one killer of women withgynecologic malignant hyperplasia. Approximately 75% of women diagnosedwith such cancers are already at an advanced stage (III and IV) of thedisease at their initial diagnosis. During the past 20 years, neitherdiagnosis nor five year survival rates have improved greatly for thesepatients. This is substantially due to the high percentage of high-stageinitial detections of the disease. Therefore, the challenge remains todevelop new markers that improve early diagnosis and thereby reduce thepercentage of high-stage initial diagnoses.

Extracellular proteases have already been implicated in the growth,spread and metastatic progression of many cancers, due to the ability ofmalignant cells not only to grow in situ, but to dissociate from theprimary tumor and to invade new surfaces. The ability to disengage fromone tissue and re-engage the surface of another tissue is what providesfor the morbidity and mortality associated with this disease. Therefore,extracellular proteases may be good candidates for markers of neoplasticdevelopment.

In order for malignant cells to grow, spread or metastasize, they musthave the capacity to invade local host tissue, dissociate or shed fromthe primary tumor, and for metastasis to occur, enter and survive in thebloodstream, implant by invasion into the surface of the target organand establish an environment conducive for new colony growth (includingthe induction of angiogenic and growth factors). During thisprogression, natural tissue barriers have to be degraded, includingbasement membranes and connective tissue. These barriers includecollagen, laminin, proteoglycans and extracellular matrix glycoproteins,including fibronectin. Degradation of these natural barriers, both thosesurrounding the primary tumor and at the sites of metastatic invasion,is believed to be brought about by the action of a matrix ofextracellular proteases.

Proteases have been classified into four families: serine proteases,metallo-proteases, aspartic proteases and cysteine proteases. Manyproteases have been shown to be involved in the human disease processand these enzymes are targets for the development of inhibitors as newtherapeutic agents. Additionally, certain individual proteases have beenshown to be induced and overexpressed in a diverse group of cancers, andas such, are potential candidates for markers of early diagnosis andpossible therapeutic intervention. A group of examples are shown inTable 1.

TABLE 1 Known proteases expressed in various cancers Gastric BrainBreast Ovarian Serine uPA uPA NES-1 NES-1 Proteases: PAI-1 PAI-1 uPA uPAtPA PAI-2 Cysteine Cathepsin B Cathepsin L Cathepsin B Cathepsin BProteases: Cathepsin L Cathepsin L Cathepsin L Metallo- Matrilysin*Matrilysin Stromelysin-3 MMP-2 proteases: Collagenase* Stromelysin MMP-8Stromelysin-1* Gelatinase B MMP-9 Gelatinase A uPA, Urokinase-typeplasminogen activator; tPA, Tissue-type plasminogen activator; PAI-I,Plasminogen activator 0 inhibitors; PAI-2, Plasminogen activatorinhibitors; NES-1, Normal epithelial cell-specific-1; MMP, Matrix Pmetallo-protease. *Overexpressed in gastrointestinal ulcers.

Significantly, there is a good body of evidence supporting thedownregulation or inhibition of individual proteases and the reductionin invasive capacity or malignancy. In work by Clark et al., inhibitionof in vitro growth of human small cell lung cancer was demonstratedusing a general serine protease inhibitor. More recently, Torres-Rosedoet al., [Proc. Natl. Acad. Sci. USA, 90, 7181-7185 (1993)] demonstratedan inhibition of hepatoma tumor cell growth using specific antisenseinhibitors for the serine protease hepsin gene. Metastatic potential ofmelanoma cells has also been shown to be reduced in a mouse model usinga synthetic inhibitor (batimastat) of metallo-proteases. Powell et al.[Cancer Research, 53, 417-422 (1993)] presented evidence to confirm thatthe expression of extracellular proteases in relatively non-invasivetumor cells enhances their malignant progression using a tumorgenic, butnon-metastatic, prostate cell line. Specifically, enhanced metastasiswas demonstrated after introducing and expressing the PUMP-1metallo-protease gene. There is also a body of data to support thenotion that expression of cell surface proteases on relativelynon-metastatic cell types increases the invasive potential of suchcells.

Thus, the prior art is deficient in a tumor marker useful as anindicator of early disease, particularly for ovarian cancers. Thepresent invention fulfills this long-standing need and desire in theart.

SUMMARY OF THE INVENTION

This invention allows for the detection of cancer, especially ovariancancer, by screening for stratum corneum chymotrytic enzyme (SCCE) mRNAin tissue, which is indicative of stratum corneum chymotrytic enzymespecifically associated with the surface of 80 percent of ovarian andother tumors. Proteases are considered to be an integral part of tumorgrowth and metastasis, and therefore, markers indicative of theirpresence or absence are useful for the diagnosis of cancer. Furthermore,the present invention is useful for treatment (i.e., by inhibiting SCCEor expression of SCCE), for targeted therapy, for vaccination, etc.

In one embodiment of the present invention, there is provided a methodof diagnosing cancer in an individual, comprising the steps of obtaininga biological sample from the individual and detecting stratum corneumchymotrytic enzyme in the sample. Usually, the presence of stratumcorneum chymotrytic enzyme in the sample is indicative of the presenceof carcinoma in the individual, and the absence of stratum corneumchymotrytic enzyme in the sample is indicative of the absence ofcarcinoma in the individual.

In another embodiment of the present invention, there is provided amethod for detecting malignant hyperplasia in a biological sample,comprising the steps of isolating mRNA from the sample and detectingstratum corneum chymotrytic enzyme mRNA in the sample. Typically, thepresence of the stratum corneum chymotrytic enzyme mRNA in the sample isindicative of the presence of malignant hyperplasia, and the absence ofthe SCCE mRNA in the sample is indicative of the absence of malignanthyperplasia.

In yet another embodiment of the present invention, there is provided amethod for detecting malignant hyperplasia in a biological sample,comprising the steps of isolating protein from the sample and detectingstratum corneum chymotrytic enzyme protein in the sample. Generally, thepresence of the SCCE protein in the sample is indicative of the presenceof malignant hyperplasia, wherein the absence of the SCCE protein in thesample is indicative of the absence of malignant hyperplasia. Thismethod may further comprise the step of comparing the SCCE protein toreference information, wherein the comparison provides a diagnosis ofthe malignant hyperplasia, or alternatively, determines a treatment ofthe malignant hyperplasia.

In still yet another embodiment of the present invention, there isprovided a method of inhibiting expression of stratum corneumchymotrytic enzyme in a cell, comprising the step of introducing avector into the cell, wherein the vector comprises a stratum corneumchymotrytic enzyme gene in opposite orientation operably linked toelements necessary for expression. Expression of the vector producesSCCE antisense mRNA in the cell, which hybridizes to endogenous SCCEmRNA and thereby inhibits expression of SCCE in the cell.

In yet another embodiment of the present invention, there is provided amethod of inhibiting stratum corneum chymotrytic enzyme protein in acell, comprising the step of introducing an antibody specific forstratum corneum chymotrytic enzyme protein or a fragment thereof intothe cell. Binding of the antibody inhibits the SCCE protein.

In another embodiment of the present invention, there is provided amethod of targeted therapy to an individual, comprising the step ofadministering a compound to an individual, wherein the compound has atargeting moiety and a therapeutic moiety, wherein the targeting moietyis specific for stratum corneum chymotrytic enzyme.

In yet another embodiment of the present invention, there is provided amethod of vaccinating an individual against stratum corneum chymotryticenzyme, comprising the steps of inoculating an individual with thestratum corneum chymotrytic enzyme protein or fragment thereof, whereinthe stratum corneum chymotrytic enzyme protein or fragment thereof lacksSCCE protease activity. Inoculation with the stratum corneum chymotryticenzyme protein or fragment thereof elicits an immune response in theindividual, thereby vaccinating the individual against stratum corneumchymotrytic enzyme.

In still another embodiment of the present invention, there is providedan oligonucleotide having a sequence complementary to SEQ ID No. 30(i.e., full length nucleotide sequence of SCCE, or fragments thereof aswould be readily recognizable to one having ordinary skill in this art).Also embodied is a composition comprising the above-describedoligonucleotide and a physiologically acceptable carrier therefore.Additionally embodied is a method of treating a neoplastic state in anindividual in need of such treatment, comprising the step ofadministering to the individual an effective dose of the above-describedoligonucleotide.

In another embodiment of the present invention, there is provided amethod of screening for compounds that inhibit stratum corneumchymotrytic enzyme activity, comprising the steps of contacting a samplewith a compound, wherein the sample comprises SCCE protein; and assayingfor SCCE protease activity. A decrease in the SCCE protease activity inthe presence of the compound relative to SCCE protease activity in theabsence of the compound is indicative of a compound that inhibitsstratum corneum chymotrytic enzyme activity.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects of the invention willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate preferred embodiments of the invention and shouldnot be considered to limit the scope of the invention.

FIG. 1 shows agarose gel comparison of PCR products derived from normaland carcinoma cDNA.

FIG. 2 shows Northern blot analysis of ovarian tumors using hepsin,SCCE, PUMP-1, TADG-14 and β-tubulin probes.

FIG. 3 shows amplification with serine protease redundant primers:histidine sense (S1) with aspartic acid antisense (AS1), using normalcDNA (Lane 1) and tumor cDNA (Lane 2); and histidine sense (S1) withserine antisense (AS2), using normal cDNA (Lane 3) and tumor cDNA (Lane4).

FIG. 4 shows amplification with cysteine protease redundant primers.Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma(Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane5).

FIG. 5 shows amplification with metallo-protease redundant primers.Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma(Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane5).

FIG. 6 shows quantitative PCR analysis of SCCE expression. Cases 3, 4and 9 are normal ovaries. Cases 19, 21, 14, 15 and 16 are LMP tumors.Cases 43, 23, 36 and 37 are ovarian carcinomas. Expression levels ofstratum corneum chymotrytic enzyme relative to β-tubulin aresignificantly elevated in tumor Cases 19, 14, 15, 16, 43, 23, 36 and 37compared to that of normal ovaries.

FIG. 7A shows Northern blot analysis of stratum corneum chymotryticenzyme mRNA from normal ovary and ovarian carcinomas. Lane 1, normalovary (case 10); Lane 2, serous carcinoma (case 35); Lane 3, mucinouscarcinoma (case 48); Lane 4, endometrioid carcinoma (case 51); and Lane5, clear cell carcinoma (case 54). Two transcripts (1.2 and 2.0 kb) weredetected in all of the subtypes of carcinoma (lanes 2-5).

FIGS. 7B and 7C show that normal human adult tissues (spleen, thymus,prostate, testis, ovary, small intestine, colon, peripheral bloodleukocyte, heart, brain, placenta, lung, liver, skeletal muscle, kidneyand pancreas) and normal human fetal tissues (brain, lung, liver andkidney) examined showed no visible SCCE transcripts.

FIG. 8 shows the ratio of SCCE expression to expression of β-tubulin innormal ovary, LMP tumor and ovarian carcinoma. SCCE mRNA expressionlevels were significantly elevated in LMP tumor (p<0.05) and carcinoma(p<0.001) compared to that in normal ovary. All 10 cases of normalovaries showed a low level of SCCE mRNA expression.

FIG. 9 shows MDA-MB-435S (Lanes 1 & 3) and HeLa (Lanes 2 & 4) celllysates were separated by SDS-PAGE and immunoblotted. Lanes 1 & 2 wereprobed with rabbit pre-immune serum as a negative control. Lanes 3 & 4were probed with polyclonal rabbit antibodies generated to peptidesderived from SCCE protein sequence.

FIG. 10A shows normal surface ovarian epithelium. Little SCCE expressionwas observed (normal ovary, X100). FIG. 10B is a negative controlsection for FIG. 10A. No nonspecific staining was observed (Normalovary, X100). FIG. 10C shows positive SCCE staining localized in thecytoplasm and the cell membrane of ovarian cancer cells (case 947, clearcell adenocarcinoma, X100). FIG. 10D is a negative control section forFIG. 10C. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100). FIG. 10E is positive stratum corneum chymotryticenzyme staining localized in the cytoplasm and the cell membrane ofovarian cancer cells. Mucin in the glands also showed positive stratumcorneum chymotrytic enzyme staining (case 947, clear celladenocarcinoma, X100). FIG. 10F is a negative control section for FIG.10E. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100).

FIG. 11A shows Northern blot analysis of hepsin expression in normalovary and ovarian carcinomas. Lane 1, normal ovary (case 10); lane 2,serous carcinoma (case 35); lane 3, mucinous carcinoma (case 48); lane4, endometrioid carcinoma (case 51); and lane 5, clear cell carcinoma(case 54). In cases 35, 51 and 54, more than a 10-fold increase in thehepsin 1.8 kb transcript abundance was observed. Northern blot analysisof hepsin in normal human fetal (FIG. 11B) and adult tissues (FIG. 11C).Significant overexpression of the hepsin transcript is noted in bothfetal liver and fetal kidney. Notably, hepsin overexpression is notobserved in normal adult tissue. Slight expression above the backgroundlevel is observed in the adult prostate.

FIG. 12A shows hepsin expression in normal (N), mucinous (M) and serous(S) low malignant potential (LMP) tumors and carcinomas (CA). FIG. 12Bshows a bar graph of expression of hepsin in 10 normal ovaries and 44ovarian carcinoma samples.

FIG. 13 shows a comparison by quantitative PCR of normal and ovariancarcinoma expression of mRNA for protease M.

FIG. 14 shows the TADG-12 catalytic domain including an insert near theHis 5′-end.

FIG. 15A shows northern blot analysis comparing TADG-14 expression innormal and ovarian carcinoma tissues. FIG. 15B shows preliminaryquantitative PCR amplification of normal and carcinoma cDNAs usingspecific primers for TADG-14.

FIG. 16A shows northern blot analysis of the PUMP-1 gene in normal ovaryand ovarian carcinomas. FIG. 16B shows northern blot analysis of thePUMP-1 gene in human fetal tissue. FIG. 16C shows northern blot analysisof the PUMP-1 gene in adult tissues.

FIG. 17A shows a comparison of PUMP-1 expression in normal and carcinomatissues using quantitative PCR with an internal β-tubulin control. FIG.17B shows the ratio of mRNA expression of PUMP-1 compared to theinternal control β-tubulin in 10 normal and 44 ovarian carcinomas.

FIG. 18 shows a comparison of Cathepsin L expression in normal andcarcinoma tissues using quantitative PCR with an internal β-tubulincontrol.

FIG. 19 is a summary of PCR amplified products for the hepsin, SCCE,protease M, PUMP-1 and Cathepsin L genes.

DETAILED DESCRIPTION OF THE INVENTION

This invention identifies that the SCCE protease on ovarian and othertumor cells is characteristic of this type of cancer, and in variouscombinations with other proteases, is characteristic of individual tumortypes. Such information can provide the basis for diagnostic tests(assays or immunohistochemistry) prognostic evaluation (depending on thedisplay pattern) and therapeutic intervention utilizing eitherantibodies directed at the protease, antisense vehicles for downregulation, or protease inhibitors both from established inhibition dataand/or for the design of new drugs. Long-term treatment of tumor growth,invasion and metastasis has not succeeded with existing chemotherapeuticagents—most tumors become resistant to drugs after multiple cycles ofchemotherapy.

A primary object of the present invention is a method for detecting thepresence of malignant hyperplasia in a tissue sample. It is an advantageof the present invention that it has as a particular object thedetection of cancer in ovarian tissue. The cancer is detected byanalyzing a biological sample for the presence of markers to proteasesthat are specific indicators of certain types of cancer cells. Thisobject may be accomplished by isolating mRNA from a sample or bydetection of proteins by polyclonal or preferably monoclonal antibodies.When using mRNA detection, the method may be carried out by combiningthe isolated mRNA with reagents to convert to cDNA according to standardmethods; treating the converted cDNA with amplification reactionreagents (such as cDNA PCR reaction reagents) in a container along withan appropriate mixture of nucleic acid primers selected from the list inTable 2 or as detailed above; reacting the contents of the container toproduce amplification products; and analyzing the amplification productsto detect the presence of malignant hyperplasia markers in the sample.For mRNA, the analyzing step may be accomplished using Northern Blotanalysis to detect the presence of malignant hyperplasia markers in theamplification product. Northern Blot analysis is known in the art. Theanalysis step may be further accomplished by quantitatively detectingthe presence of malignant hyperplasia marker in the amplificationproduce, and comparing the quantity of marker detected against a panelof expected values for known presence or absence in normal and malignanttissue derived using similar primers.

Another embodiment of the present invention are various nucleic acidsequences that are useful in the methods disclosed herein. These nucleicacid sequences are listed in Table 2. It is anticipated that thesenucleic acid sequences be used in mixtures to accomplish the utility ofthis invention. Features of such mixtures include: SEQ ID No. 1 with SEQID No. 2; SEQ ID No. 1 with SEQ ID No. 3; SEQ ID No. 4 with SEQ ID No.5; SEQ ID No. 6 with SEQ ID No. 7; SEQ ID No. 8 with SEQ ID No. 9; andSEQ ID No. 10 with SEQ ID No. 11. The skilled artisan may be able todevelop other nucleic acid sequences and mixtures thereof to accomplishthe benefit of this invention, but it is advantageous to have thesequences listed in Table 2 available without undue experimentation.

The present invention is directed toward a method of diagnosing cancerin an individual, comprising the steps of obtaining a biological samplefrom an individual and detecting stratum corneum chymotrytic enzyme inthe sample. The presence of SCCE in the sample is indicative of thepresence of cancer in the individual, wherein the absence of SCCE in thesample is indicative of the absence of cancer in the individual.Generally, detection of SCCE is by means such as Northern blot, Westernblot, PCR, dot blot, ELISA sandwich assay, radioimmunoassay, DNA arraychips and flow cytometry. An example of a typical cancer diagnosed bythis method is ovarian cancer.

The present invention is also directed toward a method for detectingmalignant hyperplasia in a biological sample, comprising the steps ofisolating mRNA from the sample; and detecting SCCE mRNA in the sample.The presence of the SCCE mRNA in the sample is indicative of thepresence of malignant hyperplasia, wherein the absence of the SCCE mRNAin the sample is indicative of the absence of malignant hyperplasia.This method may further comprise the step of comparing the SCCE mRNA toreference information, wherein the comparison provides a diagnosisand/or determines a treatment of the malignant hyperplasia. A typicalmeans of detection of stratum corneum chymotrytic enzyme mRNA is by PCRamplification, which, preferably, uses primers shown in SEQ ID No. 10and SEQ ID No. 11. Representative biological samples include a tissueand a bodily fluid, wherein the bodily fluid is preferably blood.

The present invention is additionally directed toward a method fordetecting malignant hyperplasia in a biological sample, comprising thesteps of isolating protein from the sample; and detecting stratumcorneum chymotrytic enzyme protein in the sample. The presence ofstratum corneum chymotrytic enzyme protein in the sample is indicativeof the presence of malignant hyperplasia, wherein the absence of SCCEprotein in the sample is indicative of the absence of malignanthyperplasia. This method also may comprise the step of comparing SCCEprotein to reference information, wherein the comparison provides adiagnosis or determines a treatment of the malignant hyperplasia.Preferably, the detection of SCCE protein is by immunoaffinity to anantibody which is specific for SCCE. Representative biological samplesare a tissue and a bodily fluid, and it is preferable that the bodilyfluid is blood.

The present invention is further directed toward a method of inhibitingexpression of stratum corneum chymotrytic enzyme in a cell, comprisingthe step of introducing a vector into a cell, wherein the vectorcomprises a SCCE gene in opposite orientation operably linked toelements necessary for expression, wherein expression of the vectorproduces SCCE antisense mRNA in the cell. The SCCE antisense mRNAhybridizes to endogenous SCCE mRNA, thereby inhibiting expression ofstratum corneum chymotrytic enzyme in the cell.

The present invention is still further directed toward a method ofinhibiting stratum corneum chymotrytic enzyme protein in a cell,comprising the step of introducing an antibody into a cell, wherein theantibody is specific for stratum corneum chymotrytic enzyme protein or afragment thereof. Binding of the antibody to SCCE inhibits the SCCEprotein. Preferably, the stratum corneum chymotrytic enzyme fragment isa 9-residue fragment up to a 20-residue fragment, and more preferably,the 9-residue fragment is SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86 or99.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyand a therapeutic moiety, and wherein the targeting moiety is specificfor stratum corneum chymotrytic enzyme. Preferably, the targeting moietyis an antibody specific for SCCE or a ligand or ligand binding domainthat binds SCCE. Likewise, the therapeutic moiety is preferably aradioisotope, a toxin, a chemotherapeutic agent, an immune stimulant orcytotoxic agent. Generally, the individual suffers from a disease suchas ovarian cancer, lung cancer, prostate cancer, colon cancer or anothercancer in which SCCE is overexpressed.

The present invention is additionally directed toward a method ofvaccinating an individual against stratum corneum chymotrytic enzyme,comprising the steps of inoculating an individual with SCCE protein orfragment thereof, wherein the SCCE protein or fragment thereof lacksSCCE protease activity. Inoculation with the SCCE protein, or fragmentthereof, elicits an immune response in the individual, therebyvaccinating the individual against SCCE. Preferably, the stratum corneumchymotrytic enzyme fragment is a 9-residue fragment up to a 20-residuefragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 31,32, 33, 34, 35, 36, 80, 86 or 99. Generally, this method is applicablewhen the individual has a cancer, is suspected of having a cancer or isat risk of getting a cancer.

The present invention is yet directed toward a method of producingimmune-activated cells directed toward stratum corneum chymotryticenzyme, comprising the steps of exposing dendritic cells to a SCCEprotein or fragment thereof, which lacks SCCE protease activity.Typically, exposure to the SCCE protein or fragment thereof activatesthe dendritic cells, thereby producing immune-activated cells directedtoward stratum corneum chymotrytic enzyme. Generally, theimmune-activated cells are B-cells, T-cells and/or dendrites.Preferably, the SCCE fragment is a 9-residue fragment up to a 20-residuefragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 31,32, 33, 34, 35, 36, 80, 86 or 99. Oftentimes, the dendritic cells areisolated from an individual prior to exposure and then reintroduced intothe individual subsequent to the exposure. Typically, the individual hascancer, is suspected of having cancer or is at risk of getting cancer.

The present invention is further directed toward an immunogeniccomposition, comprising an immunogenic fragment of a SCCE protein and anappropriate adjuvant. Preferably, the fragment is a 9-residue fragmentup to a 20-residue fragment, and more preferably, the 9-residue fragmentis SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86 or 99.

The present invention is further directed toward an oligonucleotidehaving a sequence complementary to SEQ ID No. 30 or a fragment thereof.The present invention further provides a composition comprising theabove-described oligonucleotide and a physiologically acceptable carriertherefore, and a method of treating a neoplastic state in an individualin need of such treatment, comprising the step of administering to theindividual an effective dose of the above-described oligonucleotide.Typically, the neoplastic state may be ovarian cancer, breast cancer,lung cancer, colon cancer, prostate cancer or another cancer in whichSCCE is overexpressed.

The present invention is still further directed toward a method ofscreening for compounds that inhibit stratum corneum chymotrytic enzymeactivity, comprising the steps of contacting a sample with a compound,wherein the sample comprises SCCE protein; and assaying for SCCEprotease activity. A decrease in the SCCE protease activity in thepresence of the compound relative to SCCE protease activity in theabsence of the compound is indicative of a compound that inhibitsstratum corneum chymotrytic enzyme activity.

The present invention is yet additionally directed toward a method fordetecting ovarian malignant hyperplasia in a biological sample,comprising the steps of isolating the proteases or protease mRNA presentin the biological sample; and detecting specific proteases or proteasemRNA present in the biological sample. The proteases are selected fromthe group consisting of hepsin, protease M, complement factor B, SCCE,cathepsin L and PUMP-1. This method may further comprise the step ofcomparing the specific proteases or protease mRNA detected to referenceinformation, wherein the comparison provides a diagnoses or determines atreatment of the malignant hyperplasia. Typically, the protease mRNA isdetected by amplification of total mRNA, and the protease is detectedwith an antibody. Representative biological samples are blood, urine,saliva, tears, interstitial fluid, ascites fluid, tumor tissue biopsyand circulating tumor cells.

It will be apparent to one skilled in the art that various substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. 1985); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. 1984); “Animal CellCulture” (R. I. Freshney, ed. 1986); “Immobilized Cells And Enzymes”(IRL Press, 1986); B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Therefore, if appearing herein, the following terms shall havethe definitions set out below.

As used herein, the term “cDNA” shall refer to the DNA copy of the mRNAtranscript of a gene.

As used herein, the term “derived amino acid sequence” shall mean theamino acid sequence determined by reading the triplet sequence ofnucleotide bases in the cDNA.

As used herein the term “screening a library” shall refer to the processof using a labeled probe to check whether, under the appropriateconditions, there is a sequence complementary to the probe present in aparticular DNA library. In addition, “screening a library” could beperformed by PCR.

As used herein, the term “PCR” refers to the polymerase chain reactionthat is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis,as well as other improvements now known in the art.

The amino acid described herein are preferred to be in the “L” isomericform. However, residues in the “D” isomeric form can be substituted forany L-amino acid residue, as long as the desired functional property ofimmunoglobulin-binding is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of apolypeptide. In keeping with standard polypeptide nomenclature, J. Biol.Chem., 243:3552-59 (1969), abbreviations for amino acid residues may beused.

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “vector” may further be defined as areplicable nucleic acid construct, e.g., a plasmid or viral nucleicacid.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single-strandedform or as a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. The structure isdiscussed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An expression vector is a replicable construct in which a nucleic acidsequence encoding a polypeptide is operably linked to suitable controlsequences capable of effecting expression of the polypeptide in a cell.The need for such control sequences will vary depending upon the cellselected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter and/or enhancer, suitablemRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing appropriate transcriptional and translational controlsignals. See, for example, techniques described in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold SpringHarbor Press, N.Y. A gene and its transcription control sequences aredefined as being “operably linked” if the transcription controlsequences effectively control transcription of the gene. Vectors of theinvention include, but are not limited to, plasmid vectors and viralvectors. Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses. In general, expression vectors contain promoter sequences whichfacilitate the efficient transcription of the inserted DNA fragment andare used in connection with a specific host. The expression vectortypically contains an origin of replication, promoter(s), terminator(s),as well as specific genes which are capable of providing phenotypicselection in transformed cells. The transformed hosts can be fermentedand cultured according to means known in the art to achieve optimal cellgrowth.

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from eukaryotic—mRNA, genomic DNA sequencesfrom eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.A polyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters typicallycontain Shine-Dalgarno ribosome-binding sequences in addition to the −10and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included near the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to enzymes, each of which cut double-stranded DNA at ornear a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into the genome of the cell. Inprokaryotes, yeast, and mammalian cells for example, the transformingDNA may be maintained on an episomal element such as a plasmid. Withrespect to eukaryotic cells, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or ancestor by mitosis. A “cell line”is a clone of a primary cell that is capable of stable growth in vitrofor many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90%or 95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example is a construct where the coding sequenceitself is not found in nature (e.g., a cDNA where the genomic codingsequence contains introns, or synthetic sequences having codonsdifferent than the native gene). Allelic variations ornaturally-occurring mutational events do not give rise to a heterologousregion of DNA as defined herein.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Proteins canalso be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. The preferred isotope may be selected from ³H, ¹⁴C,³²P, ³⁵S ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090,3,850,752, and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system developed and utilized in the art is known asa receptor assay. In a receptor assay, the material to be assayed isappropriately labeled and then certain cellular test colonies areinoculated with a quantitiy of both the label after which bindingstudies are conducted to determine the extent to which the labeledmaterial binds to the cell receptors. In this way, differences inaffinity between materials can be ascertained.

An assay useful in the art is known as a “cis/trans” assay. Briefly,this assay employs two genetic constructs, one of which is typically aplasmid that continually expresses a particular receptor of interestwhen transfected into an appropriate cell line, and the second of whichis a plasmid that expresses a reporter such as luciferase, under thecontrol of a receptor/ligand complex. Thus, for example, if it isdesired to evaluate a compound as a ligand for a particular receptor,one of the plasmids would be a construct that results in expression ofthe receptor in the chosen cell line, while the second plasmid wouldpossess a promoter linked to the luciferase gene in which the responseelement to the particular receptor is inserted. If the compound undertest is an agonist for the receptor, the ligand will complex with thereceptor, and the resulting complex will bind the response element andinitiate transcription of the luciferase gene. The resultingchemiluminescence is then measured photometrically, and dose responsecurves are obtained and compared to those of known ligands. Theforegoing protocol is described in detail in U.S. Pat. No. 4,981,784.

As used herein, the term “host” is meant to include not only prokaryotesbut also eukaryotes such as yeast, plant and animal cells. A recombinantDNA molecule or gene which encodes a human SCCE protein of the presentinvention can be used to transform a host using any of the techniquescommonly known to those of ordinary skill in the art. Especiallypreferred is the use of a vector containing coding sequences for thegene which encodes a human SCCE protein of the present invention forpurposes of prokaryote transformation. Prokaryotic hosts may include E.coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cellsand insect cells.

As used herein, “substantially pure DNA” means DNA that is not part of amilieu in which the DNA naturally occurs, by virtue of separation(partial or total purification) of some or all of the molecules of thatmilieu, or by virtue of alteration of sequences that flank the claimedDNA. The term therefore includes, for example, a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein. Also included is arecombinant DNA which includes a portion of the nucleotides listed inSEQ ID No. 30 and which encodes an alternative splice variant of stratumcorneum chymotrytic enzyme.

By a “substantially pure protein” is meant a protein which has beenseparated from at least some of those components which naturallyaccompany it. Typically, the protein is substantially pure when it is atleast 60% (by weight) free from the proteins and othernaturally-occurring organic molecules with which it is naturallyassociated in vivo. Preferably, the purity of the preparation (byweight) is at least 75%, more preferably at least 90%. and mostpreferably at least 99%. A substantially pure SCCE protein may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding a SCCE polypeptide; orby chemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., column chromatography, such as immunoaffinitychromatography using an antibody specific for SCCE, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein is substantially free ofnaturally associated components when it is separated from at least someof those contaminants which accompany it in its natural state. Thus, aprotein which is chemically synthesized or produced in a cellular systemdifferent from the cell from which it naturally originates will be, bydefinition, substantially free from its naturally associated components.Accordingly, substantially pure proteins include eukaryotic proteinssynthesized in E. coli, other prokaryotes, or any other organism inwhich they do not naturally occur.

The term “oligonucleotide”, as used herein, is defined as a moleculecomprised of two or more ribonucleotides, preferably more than three.Its exact size will depend upon many factors, which, in turn, dependupon the ultimate function and use of the oligonucleotide. The term“primer”, as used herein, refers to an oligonucleotide, whetheroccurring naturally (as in a purified restriction digest) or producedsynthetically, and which is capable of initiating synthesis of a strandcomplementary to a nucleic acid when placed under appropriateconditions, i.e., in the presence of nucleotides and an inducing agent,such as a DNA polymerase, and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, sequenceand/or homology of primer and the method used. For example, indiagnostic applications, the oligonucleotide primer typically contains15-25 or more nucleotides, depending upon the complexity of the targetsequence, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary toparticular target DNA sequences. This means that the primers must besufficiently complementary to hybridize with their respective strands.Therefore, the primer sequence need not reflect the exact sequence ofthe template. For example, a non-complementary nucleotide fragment(i.e., containing a restriction site) may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementary with the sequence tohybridize therewith and form the template for synthesis of the extensionproduct.

The probe to which the DNA of the invention hybridizes preferablyconsists of a sequence of at least 20 consecutive nucleotides, morepreferably 40 nucleotides, even more preferably 50 nucleotides, and mostpreferably 100 nucleotides or more (up to 100%) of the coding sequenceof the nucleotides listed in SEQ ID No. 30 or the complement thereof.Such a probe is useful for detecting expression of SCCE in a cell by amethod including the steps of (a) contacting mRNA obtained from the cellwith a labeled SCCE hybridization probe; and (b) detecting hybridizationof the probe with the mRNA.

By “high stringency” is meant DNA hybridization and wash conditionscharacterized by high temperature and low salt concentration, e.g., washconditions of 65° C. at a salt concentration of approximately 0.1× SSC,or the functional equivalent thereof. For example, high stringencyconditions may include hybridization at about 42° C. in the presence ofabout 50% formamide; a first wash at about 65° C. with about 2× SSCcontaining 1% SDS; followed by a second wash at about 65° C. with about0.1× SSC.

The DNA may have at least about 70% sequence identity to the codingsequence of the nucleotides listed in SEQ ID No. 30, preferably at least75% (e.g., at least 80%); and most preferably at least 90%. The identitybetween two sequences is a direct function of the number of matching oridentical positions. When a position in both of the two sequences isoccupied by the same monomeric subunit, e.g., if a given position isoccupied by an adenine in each of two DNA molecules, then they areidentical at that position. For example, if 7 positions in a sequence 10nucleotides in length are identical to the corresponding positions in asecond 10-nucleotide sequence, then the two sequences have 70% sequenceidentity. The length of comparison sequences will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 100 nucleotides. Sequenceidentity is typically measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group (GCG),University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705).

The present invention comprises a vector comprising a DNA sequence whichencodes a SCCE protein, wherein said vector is capable of replication ina host, and comprises, in operable linkage: a) an origin of replication;b) a promoter; and c) a DNA sequence coding for the SCCE protein.Preferably, the vector of the present invention contains a portion ofthe DNA sequence shown in SEQ ID No. 30. Vectors may be used to amplifyand/or express nucleic acid encoding a SCCE protein or fragment thereof.

In addition to substantially full-length proteins, the invention alsoincludes fragments (e.g., antigenic fragments) of the SCCE protein. Asused herein, “fragment,” as applied to a polypeptide, will ordinarily beat least 10 residues, more typically at least 20 residues, andpreferably at least 30 (e.g., 50) residues in length, but less than theentire, intact sequence. Fragments of the SCCE protein can be generatedby methods known to those skilled in the art, e.g., by enzymaticdigestion of naturally occurring or recombinant SCCE protein, byrecombinant DNA techniques using an expression vector that encodes adefined fragment of SCCE, or by chemical synthesis. The ability of acandidate fragment to exhibit a characteristic of SCCE (e.g., binding toan antibody specific for SCCE) can be assessed by methods describedherein. Purified SCCE or antigenic fragments of SCCE can be used togenerate new antibodies or to test existing antibodies (e.g., aspositive controls in a diagnostic assay) by employing standard protocolsknown to those skilled in the art. Included in this invention ispolyclonal antisera generated by using SCCE or a fragment of SCCE as theimmunogen in, e.g., rabbits. Standard protocols for monoclonal andpolyclonal antibody production known to those skilled in this art areemployed. The monoclonal antibodies generated by this procedure can bescreened for the ability to identify recombinant SCCE cDNA clones, andto distinguish them from other cDNA clones.

Further included in this invention are SCCE proteins which are encoded,at least in part, by portions of SEQ ID No. 29, e.g., products ofalternative mRNA splicing or alternative protein processing events, orin which a section of SCCE sequence has been deleted. The fragment, orthe intact SCCE polypeptide, may be covalently linked to anotherpolypeptide, e.g., one which acts as a label, a ligand or a means toincrease antigenicity.

The invention also includes a polyclonal or monoclonal antibody whichspecifically binds to stratum corneum chymotrytic enzyme. The inventionencompasses not only an intact monoclonal antibody, but also animmunologically-active antibody fragment, e.g., a Fab or (Fab)₂fragment; an engineered single chain Fv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

In one embodiment, the antibody, or a fragment thereof, may be linked toa toxin or to a detectable label, e.g., a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label, or calorimetric label. Examplesof suitable toxins include diphtheria toxin, Pseudomonas exotoxin A,ricin, and cholera toxin. Examples of suitable enzyme labels includemalate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase,etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986)Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16, 93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examplesof suitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an ophthaldehyde label, afluorescamine label, etc. Examples of chemiluminescent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,an imidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known and used by thoseof ordinary skill in the art. Typical techniques are described byKennedy et al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al.,(1977) Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in thelatter are the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide estermethod. All of these methods are incorporated by reference herein.

Also within the invention is a method of detecting SCCE protein in abiological sample, which includes the steps of contacting the samplewith the labeled antibody, e.g., radioactively tagged antibody specificfor stratum corneum chymotrytic enzyme, and determining whether theantibody binds to a component of the sample. Antibodies to the SCCEprotein can be used in an immunoassay to detect increased levels of SCCEprotein expression in tissues suspected of neoplastic transformation.These same uses can be achieved with Northern blot assays and analyses.

As described herein, the invention provides a number of diagnosticadvantages and uses. For example, the SCCE protein is useful indiagnosing cancer in different tissues since this protein is highlyoverexpressed in tumor cells. Antibodies (or antigen-binding fragmentsthereof) which bind to an epitope specific for SCCE are useful in amethod of detecting SCCE protein in a biological sample for diagnosis ofcancerous or neoplastic transformation. This method includes the stepsof obtaining a biological sample (e.g., cells, blood, plasma, tissue,etc.) from a patient suspected of having cancer, contacting the samplewith a labeled antibody (e.g., radioactively tagged antibody) specificfor SCCE, and detecting the SCCE protein using standard immunoassaytechniques such as an ELISA. Antibody binding to the biological sampleindicates that the sample contains a component which specifically bindsto an epitope within stratum corneum chymotrytic enzyme.

Likewise, a standard Northern blot assay can be used to ascertain therelative amounts of SCCE mRNA in a cell or tissue obtained from apatient suspected of having cancer, in accordance with conventionalNorthern hybridization techniques known to those of ordinary skill inthe art. This Northern assay uses a hybridization probe, e.g.,radiolabelled SCCE cDNA, either containing the full-length, singlestranded DNA having a sequence complementary to SEQ ID No. 30, or afragment of that DNA sequence at least 20 (preferably at least 30, morepreferably at least 50, and most preferably at least 100 consecutivenucleotides in length). The DNA hybridization probe can be labeled byany of the many different methods known to those skilled in this art.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion:

EXAMPLE 1

Amplification of Serine Proteases Using Redundant and Specific Primers

Only cDNA preparations deemed free of genomic DNA were used for geneexpression analysis. Redundant primers were prepared for serineproteases, metallo-proteases and cysteine protease. The primers weresynthesized to consensus sequences of amino acid surrounding thecatalytic triad for serine proteases, viz. histidine . . . aspartate . .. and serine. The sequences of both sense (histidine & aspartate) andantisense (aspartate and serine) redundant primers are shown in Table 2.

TABLE 2 PCR Primers 5′→3′ SEQ ID No. Redundant Primers: Serine Protease(histidine) = S1 tgggtigtiacigcigcica(ct)tg 1 Serine Protease (asparticacid) = AS1 a(ag)ia(ag)igciatitcitticc 2 Serine Protease (serine) = AS11a(ag)iggiccicci(cg)(ta)(ag)tcicc 3 Cysteine Protease - senseca(ag)ggica(ag)tg(ct)ggi(ta)(cg)itg(ct)tgg 4 Cysteine Protease -antisense taiccicc(ag)tt(ag)caicc(ct)tc 5 Metallo Protease - sensecci(ac)gitg(tc)ggi(ga)(ta)icciga 6 Metallo Protease - antisensett(ag)tgicciai(ct)tc(ag)tg 7 Specific Primers: Serine Protease (hepsin)= sense tgtcccgatggcgagtgttt 8 Serine Protease (hepsin) = antisensecctgttggccatagtactgc 9 Serine Protease (SCCE) = senseagatgaatgagtacaccgtg 10 Serine Protease (SCCE) = antisenseccagtaagtccttgtaaacc 11 Serine Protease (Comp B) = senseaagggacacgagagctgtat 12 Serine Protease (Comp B) = antisenseaagtggtagttggaggaagc 13 Serine Protease (Protease M) = sensectgtgatccaccctgactat 20 Serine Protease (Protease M) = antisensecaggtggatgtatgcacact 21 Serine Protease (TADG12) = sense (Ser10-s)gcgcactgtgtttatgagat 22 Serine Protease (TADG12) = antisense (Ser10-as)ctctttggcttgtacttgct 23 Serine Protease (TADG13) = sensetgagggacatcattatgcac 24 Serine Protease (TADG13) = antisensecaagttttccccataattgg 25 Serine Protease (TADG14) = senseacagtacgcctgggagacca 26 Serine Protease (TADG14) = antisensectgagacggtgcaattctgg 27 Cysteine Protease (Cath-L) = senseattggagagagaaaggctac 14 Cysteine Protease (Cath-L) = antisensecttgggattgtacttacagg 15 Metallo Protease (PUMP1) = sensecttccaaagtggtcacctac 16 Metallo Protease (PUMP1) = antisensectagactgctaccatccgtc 17

EXAMPLE 2

Carcinoma Tissue

Several protease entities were identified and subcloned from PCRamplification of cDNA derived from serous cystadenocarcinomas.Therefore, the proteases described herein are reflective of surfaceactivities for this type of carcinoma, the most common form of ovariancancer. It was also shown that PCR amplification bands unique to themucinous tumor type and the clear cell type have similar base pair size.About 20-25% of ovarian cancers are classified as either mucinous, clearcell, or endometrioid.

EXAMPLE 3

Ligation Transformation and Sequencing

To determine the identity of the PCR products, all the appropriate bandswere ligated into Promega T-vector plasmid and the ligation product wasused to transform JM109 cells (Promega) grown on selective media. Afterselection and culturing of individual colonies, plasmid DNA was isolatedby means of the WIZARD MINIPREP™ DNA purification system (Promega).Inserts were sequenced using a Prism Ready Reaction Dydeoxy Terminatorscycle sequencing kit (Applied Biosystems). Residual dye terminators wereremoved from the completed sequencing reaction using a CENTRISEP SPIN™column (Princeton Separation), and samples were loaded into an AppliedBiosystems Model 373A DNA sequencing system. The results of subcloningand sequencing for the serine protease primers are summarized in Table3.

TABLE 3 Serine Protease Candidates Subclone Primer Set Gene Candidate 1His-Ser Hepsin 2 His-Ser SCCE 3 His-Ser Compliment B 4 His-Asp Cofactor1 5 His-Asp TADG-12* 6 His-Ser TADG-13* 7 His-Ser TADG-14* 8 His-SerProtease M 9 His-Ser TADG-15* *indicates novel proteases

EXAMPLE 4

Cloning and Characterization

Cloning and characterization of new gene candidates was undertaken toexpand the panel representative of extracellular proteases specific forovarian carcinoma subtypes. Sequencing of the PCR products derived fromtumor cDNA confirms the potential candidacy of these genes. The threenovel genes all have conserved residues within the catalytic triadsequence consistent with their membership in the serine protease family.

PCR products amplified from normal and carcinoma cDNAs were comparedusing sense-histidine and antisense-aspartate as well as sense-histidineand antisense-serine. The anticipated PCR products of approximately 200bp and 500 bp for those pairs of primers were observed (aspartate isapproximately 50-70 amino acids downstream from histidine, and serine isabout 100-150 amino acids toward the carboxy end from histidine).

FIG. 1 shows a comparison of PCR products derived from normal andcarcinoma cDNA as shown by staining in an agarose gel. Two distinctbands in Lane 2 were present in the primer pair sense-His/antisense ASP(AS1) and multiple bands of about 500 bp are noted in the carcinoma lanefor the sense-His/antisense-Ser (AS2) primer pairs in Lane 4.

EXAMPLE 5

Quantitative PCR

The mRNA overexpression of SCCE was detected and determined usingquantitative PCR. Quantitative PCR was performed generally according tothe method of Noonan et al. [Proc. Natl. Acad. Sci., USA, 87:7160-7164(1990)]. The following oligonucleotide primers were used:

SCCE:

forward 5′-AGATGAATGAGTACACCGTG-3′ (SEQ ID No.10), and reverse5′-CCAGTAAGTCCTTGTAAACC-3′ (SEQ ID No.11); and β-tubulin: forward5′-TGCATTGACAACGAGGC -3′ (SEQ ID No.18), and reverse 5′-CTGTCTTGACATTGTTG -3′ (SEQ ID No.19).β-tubulin was utilized as an internal control. The predicted sizes ofthe amplified genes were 339 bp for SCCEand 454 bp for β-tubulin. Theprimer sequences used in this study were designed according to the cDNAsequences described by Hansson et al. [J. Biol. Chem., 269, 19420-19426(1994)] for SCCE, and Hall et al. [Mol. Cell. Biol., 3, 854-862 (1983)]for β-tubulin. The PCR reaction mixture consisted of cDNA derived from50 ng of mRNA converted by conventional techniques, 5 pmol of sense andantisense primers for both the SCCE gene and the β-tubulin gene, 200μmol of dNTPs, 5 μCi of α-³²PdCTP and 0.25 units of Taq DNA polymerasewith reaction buffer (Promega) in a final volume of 25 μl. The targetsequences were amplified in parallel with the β-tubulin gene. Thirtycycles of PCR were carried out in a Thermal Cycler (Perkin-Elmer Cetus).Each cycle of PCR included 30 sec of denaturation at 95° C., 30 sec ofannealing at 63° C. and 30 sec of extension at 72° C. It was previouslyestablished and confirmed for SCCE that co-amplification with β-tubulinunder these conditions for 30 cycles remain linear for both products.

The PCR products were separated on 2% agarose gels and the radioactivityof each PCR product was determined by using a Phospho Imager (MolecularDynamics). In the present study, expression of SCCE was calculated asthe ratio (SCCE/β-tubulin) as measured by phosphoimager. Theoverexpression cut-off value was defined as the mean value for normalovary +2SD. The student's t test was used for the comparison of the meanvalues of normal ovary and tumors.

Experiments comparing PCR amplification in normal ovary and ovariancarcinoma suggested overexpression and/or alteration in mRNA transcriptin tumor tissues. Northern blot analysis of TADG-14 confirms atranscript size of 1.4 kb and data indicate overexpression in ovariancarcinoma (FIG. 2). Isolation and purification using both PCR and aspecific 250 bp PCR product to screen positive plaques yielded a 1.2 kbclone of TADG-14. Other proteases were amplified by the same methodusing the appropriate primers from Table 2.

EXAMPLE 6

Tissue Bank

A tumor tissue bank of fresh frozen tissue of ovarian carcinomas asshown in Table 4 was used for evaluation. Approximately 100 normalovaries removed for medical reasons other than malignancy were obtainedfrom surgery and were available as controls.

TABLE 4 Ovarian Cancer Tissue Bank Total Stage I/11 Stage III/IV NoStage Serous Malignant 166 15 140 8 LMP 16 9 7 0 Benign 12 0 0 12Mucinous Malignant 26 6 14 6 LMP 28 25 3 0 Benign 3 0 0 3 EndometrioidMalignant 38 17 21 0 LMP 2 2 0 0 Benign 0 0 0 0 Other* Malignant 61 2329 9 LMP 0 0 0 0 Benign 5 0 0 5 *Other category includes the followingtumor types: Brenner's tumor, thecoma, teratoma, fibrothecoma, fibroma,granulosa cell, clear cell, germ cell, mixed mullerian, stromal,undifferentiated, and dysgerminoma.

From the tumor bank, approximately 100 carcinomas were evaluatedencompassing most histological sub-types of ovarian carcinoma, includingborderline or low-malignant potential tumors and overt carcinomas. Theapproach included using mRNA prepared from fresh frozen tissue (bothnormal and malignant) to compare expression of genes in normal, lowmalignant potential tumors and overt carcinomas. The cDNA prepared frompolyA+ mRNA was deemed to be genomic DNA free by checking allpreparations with primers that encompassed a known intron-exon splicesite using both β-tubulin and p53 primers.

EXAMPLE 7

Northern Blots

Significant information can be obtained by examining the expression ofthese candidate genes by Northern blot. Analysis of normal adultmulti-tissue blots offers the opportunity to identify normal tissueswhich may express the protease. Ultimately, if strategies for inhibitionof proteases for therapeutic intervention are to be developed, it isessential to appreciate the expression of these genes in normal tissueif and when it occurs.

Northern panels for examining expression of genes in a multi-tissuenormal adult as well as fetal tissue are commercially available(CLONTECH). Such evaluation tools are not only important to confirm theoverexpression of individual transcripts in tumor versus normal tissues,but also provides the opportunity to confirm transcript size, and todetermine if alternate splicing or other transcript alteration may occurin ovarian carcinoma.

EXAMPLE 8

Northern Blot Analysis

Northern blot analysis was performed as follows: 10 μg of mRNA wasloaded onto a 1% formaldehyde-agarose gel, electrophoresed and blottedonto a HyBond-N⁻™ nylon membrane (Amersham). ³²P-labeled cDNA probeswere made using Prime-a-Gene Labeling System™ (Promega). The PCRproducts amplified by specific primers were used as probes. Blots wereprehybridized for 30 min and then hybridized for 60 min at 68° C. with³²P-labeled cDNA probe in ExpressHyb™ Hybridization Solution (CLONTECH).Control hybridization to determine relative gel loading was accomplishedusing the β-tubulin probe.

Normal human tissues including spleen, thymus, prostate, testis, ovary,small intestine, colon, peripheral blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas and normalhuman fetal tissues; brain, lung, liver and kidney (Human MultipleTissue Northern Blot; CLONTECH) were all examined using the samehybridization procedure.

EXAMPLE 9

PCR Products Corresponding to Serine, Cysteine and Metallo-Proteases

Based on their unique expression in either low malignant potentialtumors or carcinomas, PCR-amplified cDNA products were cloned andsequenced and the appropriate gene identified based upon nucleotide andamino acid sequences stored in the GCG and EST databases. FIGS. 3, 4 & 5show the PCR product displays comparing normal and carcinomatous tissuesusing redundant primers for serine proteases (FIG. 3), for cysteineproteases (FIG. 4) and for metallo-proteases (FIG. 5). Note thedifferential expression in the carcinoma tissues versus the normaltissues. The proteases were identified using redundant cDNA primers (seeTable 2) directed towards conserved sequences that are associated withintrinsic enzyme activity (for serine proteases, cysteine proteases andmetallo-proteases) by comparing mRNA expression in normal, low malignantpotential and overt ovarian carcinoma tissues according to Sakanari etal. [Biochemistry 86, 4863-4867 (1989)].

EXAMPLE 10

Serine Proteases

For the serine protease group, using the histidine domain primer sense,S1, in combination with antisense primer AS2, the following proteaseswere identified:

(a) Hepsin, a trypsin-like serine protease cloned from hepatoma cellsshown to be a cell surface protease essential for the growth of hepatomacells in culture and highly expressed in hepatoma tumor cells (FIG. 3,Lane 4);

(b) Complement factor B protease (human factor IX), a protease involvedin the coagulation cascade and associated with the production andaccumulation of fibrin split products associated with tumor cells (FIG.3, Lane 4). Compliment factor B belongs in the family of coagulationfactors X (Christmas factor). As part of the intrinsic pathway,compliment factor B catalyzes the proteolytic activation of coagulationfactor X in the presence of Ca²⁺ phospholipid and factor VIIIa e5; and

(c) A stratum corneum chymotryptic enzyme (SCCE) serine proteaseinvolved in desquarnation of skin cells from the human stratum corneum(FIG. 3, Lane 4). SCCE is expressed in keratinocytes of the epidermisand functions to degrade the cohesive structures in the cornified layerto allow continuous skin surface shedding.

EXAMPLE 11

Cysteine Proteases

In the cysteine protease group, using redundant sense and anti-senseprimers for cysteine proteases, one unique PCR product was identified byoverexpression in ovarian carcinoma when compared to normal ovariantissue (FIG. 4, Lanes 3-5). Cloning and sequencing this PCR productidentified a sequence of Cathepsin L, which is a lysomal cysteineprotease whose expression and secretion is induced by malignanttransformation, growth factors and tumor promoters. Many human tumors(including ovarian) express high levels of Cathepsin L. Cathepsin Lcysteine protease belongs in the stromolysin family and has potentelastase and collagenase activities. Published data indicates increasedlevels in the serum of patients with mucinous cystadenocarcinoma of theovary. It has not heretofore been shown to be expressed in other ovariantumors.

EXAMPLE 12

Metallo-Proteases

Using redundant sense and anti-sense primers for the metallo-proteasegroup, one unique PCR product was detected in the tumor tissue which wasabsent in normal ovarian tissue (FIG. 5, Lanes 2-5). Subcloning andsequencing this product indicates it has complete homology in theappropriate region with the so-called PUMP-1 (MMP-7) gene. Thiszinc-binding metallo-protease is expressed as a proenzyme with a signalsequence and is active in gelatin and collagenase digestion. PUMP-1 hasalso been shown to be induced and overexpressed in 9 of 10 colorectalcarcinomas compared to normal colon tissue, suggesting a role for thissubstrate in the progression of this disease.

EXAMPLE 13

mRNA Expression of SCCE in Ovarian Tumors

To evaluate mRNA expression of SCCE in ovarian tumors, semi-quantitativePCR was performed. A preliminary study confirmed the linearity of thePCR amplification (Shigemasa et al., J. Soc Gynecol Invest 4, 95-102,1997; Hall et al., Mol Cell Biol 3, 854-862, 1983). FIG. 6 shows anexample of comparative PCR using SCCE primers co-amplified with theinternal control β-tubulin primers. Analysis of the data as measuredusing the phosphoimager and compared as ratios of expression(SCCE/β-tubulin) indicate that SCCE expression is elevated in tumorcases 19, 14, 15, 16, 43, 23, 36 and 37 compared to that of normalovaries.

To confirm the results of the initial quantitative PCR and to examinethe size of the transcript, Northern blot hybridization was performed inrepresentative cases of each histological type of carcinoma (FIG. 7A).Northern blot hybridization with a ³²P-labeled SCCE probe (nucleotides232-570) revealed 1.2 kb and 2.0 kb transcripts, as reported previouslyin normal skin tissue (Hansson et al., J. Biol Chem 269, 19420-19426,1994). Those tumor cases which showed overexpression of SCCE byquantitative PCR also showed intense bands of SCCE transcript expressionby Northern blot analysis including serous, mucinous, endometrioid andclear cell carcinoma. No transcripts were detected in normal ovariantissue (Lane 1). Normal human tissues (spleen, thymus, prostate, testis,ovary, small intestine, colon, peripheral blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas) and normalhuman fetal tissues (brain, lung, liver and kidney) examined by Northernblot analysis showed no visible SCCE transcripts (FIGS. 7B & 7C). Blotsfor normal human adult tissues and fetal tissues were subsequentlyprobed to confirm the presence of β-tubulin transcripts.

Table 5 summarizes the results of the evaluation of SCCE expression in10 individual normal ovarian tissues and 44 ovarian carcinomas. Overall,SCCE mRNA overexpression (overexpression=mean value for normalovary+2SD) was found in 8 of 12 LMP tumors (66.7%) and 25 of 32carcinoma cases (78.1%) with p values of <0.05 and <0.001 respectively(FIG. 8). Overexpression of SCCE transcripts was detected in all ovariancarcinoma subtypes and in both early stage and late stage tumor samples.In the five cases where positive confirmation of lymph node metastasiswas identified, all five cases showed overexpression of SCCE at a levelof more than four standard deviations above the level for normal ovary.It should be noted that three of these tumors were classified as lowmalignant potential tumors (all serous adenomas) suggesting a possiblerelationship between the progression of early stage disease to the lymphwhen overexpression of SCCE is manifest.

TABLE 5 Patient Characteristics and Expression of SCCE Gene mRNAexpression^(c) Case Histological Type^(a) Stage/Grade LN^(b) SCCE 1normal ovary n 2 normal ovary n 3 normal ovary n 4 normal ovary n 5normal ovary n 6 normal ovary n 7 normal ovary n 8 normal ovary n 9normal ovary n 10 normal ovary n 11 s adenoma (LMP) 1/1 n 4+ 12 sadenoma (LMP) 1/1 NE n 13 s adenoma (LMP) 1/1 NE 2+ 14 s adenoma (LMP)1/1 n 4+ 15 s adenoma (LMP) 3/1 p 4+ 16 s adenoma (LMP) 3/1 p 4+ 17 sadenoma (LMP) 3/1 p 4+ 18 m adenoma (LMP) 1/1 NE 4+ 19 m adenoma (LMP)1/1 n 4+ 20 m adenoma (LMP) 1/1 n n 21 m adenoma (LMP) 1/1 NE n 22 madenoma (LMP) 1/1 NE n 23 s carcinoma 1/2 n 4+ 24 s carcinoma 1/3 n 4+25 s carcinoma 3/1 NE 4+ 26 s carcinoma 3/2 NE 4+ 27 s carcinoma 3/2 p4+ 28 s carcinoma 3/2 NE 4+ 29 s carcinoma 3/3 NE 4+ 30 s carcinoma 3/3NE 4+ 31 s carcinoma 3/3 NE 4+ 32 s carcinoma 3/3 NE 4+ 33 s carcinoma3/3 n 4+ 34 s carcinoma 3/3 NE n 35 s carcinoma 3/3 NE 4+ 36 s carcinoma3/3 NE 4+ 37 s carcinoma 3/3 NE 4+ 38 s carcinoma 3/3 n 4+ 39 scarcinoma 3/2 NE 4+ 40 s carcinoma 3/3 NE 4+ 41 s carcinoma 3/2 NE n 42m carcinoma 1/2 n n 43 m carcinoma 2/2 NE 4+ 44 m carcinoma 2/2 n n 45 mcarcinoma 3/1 NE n 46 m carcinoma 3/2 NE n 47 m carcinoma 3/2 NE n 48 mcarcinoma 3/3 NE 4+ 49 e carcinoma 2/3 n 4+ 50 e carcinoma 3/2 NE 4+ 51e carcinoma 3/3 NE 4+ 52 c carcinoma 1/3 n 4+ 53 c carcinoma 1/1 n 4+ 54c carcinoma 3/2 p 4+ ^(a)s; serous, m; mucinous, e; endometrioid, c;clear cell; ^(b)LN; lymph node metastasis, p; positive, n; negative, NE;not examined; ^(c)n, normal range is equal to Mean ± 2SD, 2+; Mean + 2SDto + 4SD, 4+; Mean + 4SD or greater

The expression ratio (mean value±SD) for normal ovary was determined as0.046±0.023, for LMP tumors as 0.405±0.468 and for carcinoma as0.532±0.824 (Table 6). From a histological point of view, overexpressionof SCCE was observed in 23 of 26 serous tumors (88.5%) including 6 of 7LMP tumors and 17 of 19 carcinomas. However only 4 of 12 mucinous tumors(33.3%) including 2 of 5 LMP tumors and 2 of 7 carcinomas showedoverexpression of SCCE. For endometrioid and clear cell carcinoma,stratum corneum chymotrytic enzyme was found to be overexpressed in all6 cases (Table 6).

TABLE 6 Overexpression of SCCE in Ovarian Carcinoma Overexpression of NSCCE Expression Ratio^(a) Normal 10  0 (0%) 0.046 ± 0.023 LMP 12  8(66.7%) 0.405 ± 0.468 serous 7 6 (85.7%) 0.615 ± 0.518 mucinous 5 2(40.0%) 0.111 ± 0.117 Carcinoma 32  25 (78.1%) 0.532 ± 0.824 serous 19 17 (89.5%) 0.686 ± 1.027 mucinous 7 2 (28.6%) 0.132 ± 0.265 endometrioid3 3 (100%) 0.511 ± 0.205 clear cell 3 3 (100%) 0.515 ± 0.007 ^(a)Theratio of expression level of SCCE to β-tubulin (mean ± SD)

EXAMPLE 14

Western Blot

Polyclonal rabbit antibodies were generated by immunization with acombination of 2 poly-lysine linked multiple Ag peptides derived fromSCCE protein sequences PLQILLLSLALE (SEQ ID No. 28) and SFRHPGYSTQTH(SEQ ID No. 29). Approximately 20 ng of MDA-MBA-435S and HeLa celllysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDFat 100 V for 40 minutes at 4° C. The proteins were fixed to the membraneby incubation in 50% MeOH for 10 minutes. The membrane was blockedovernight in TBS, pH 7.8 containing 0.2% non-fat milk. Primary antibodywas added to the membrane at a dilution of 1:100 in 0.2% milk/TBS andincubated for 2 hours at room temperature. The blot was washed andincubated with a 1:3000 dilution of alkaline-phosphatase conjugated goatanti-rabbit IgG (BioRad) for one hour at room temperature. The blot waswashed and incubated with a chemiluminescent substrate before a 10second exposure to X-ray film for visualization.

Two cell lines HeLa and MDA-MB-435S previously shown to express mRNAtranscripts were examined by Western blot to confirm the presence ofSCCE protein. FIG. 9 indicates that polyclonal antibodies developed topeptides (12 mers bound to polylysine) derived from the amino andcarboxy termini of SCCE bind a protein of approximately 30 kDa incytosolic extracts of HeLa and MDA-MB-435S cells. The ovarian tumor cellline CAOV3 was also examined for SCCE expression and a protein productcould not be detected (data not shown). This molecular size proteinagrees with the anticipated and known parameters for the SCCE protein.It should be noted that only a single band was detected by Western blotanalysis of cystosolic protein. It might be anticipated that the SCCEprotein prior to secretion would be present in the inactivated parentform i.e. the seven amino terminal peptide removed on activation wouldstill be present on the enzyme. In this pre-active form of the enzyme itwould be anticipated that the apparent molecular weight on Western blotwould be about 30 kDa.

EXAMPLE 15

Immunohistochemistry

Immunohistochemical localization of SCCE antigen was examined usingnormal ovaries, mucinous LMP tumor and adenocarcinomas (including serousadenocarcinomas, mucinous adenocarcinoma and clear cell carcinomas) inthe same series of the samples for mRNA isolation. Formalin fixed andparaffin embedded sections, 4 μm thick, were cut and mounted onaminopropyltriethoxysilane treated slides. Slides were routinelydeparaffinized with xylene and rehydrated with a series of ethanolwashes. Nonenzymatic antigen retrieval was performed by processing usingmicrowave heat treatment in 0.01 M sodium citrate buffer (pH 6.0).Immunohistochemical staining was performed manually using theavidin-biotin peroxidase complex technique (Vectastain Elite ABC kit,Vector Laboratories). Anti-SCCE rabbit polyclonal antibody was generatedby immunization with a combination of 2 poly-lysine linked multiple Agpeptide derived from the SCCE protein-sequences.

This indirect immunoperoxidase staining procedure was performed at roomtemperature. Endogenous peroxidase and nonspecific background stainingwere blocked by incubating slides with methanol with 0.3% H₂O₂ for 30minutes. After washing with phosphate-buffered saline (PBS) for 10minutes, sections were incubated with biotinylated anti-rabbit IgG for30 minutes. After washing with PBS for 10 minutes, slides were incubatedwith ABC reagent for 30 minutes. The final products were visualized byusing AEC substrate system (DAKO Corporation) and sections werecounterstained with Mayer hematoxylin for 20 seconds before mounting.Positive controls and negative controls were used for each section.Negative controls were performed by using normal rabbit serum instead ofthe primary antibody. All experiments were duplicated. The stainedslides were examined microscopically by 3 observers. More than 10% ofpositive tumor cells was the criterion for a 1+ positive staining andmore than 50% of positive tumor cells was the criterion for a 2+positive staining.

To further confirm the presence of the SCCE protein in ovarian tumorcells as opposed to its elaboration by supporting stromal or bloodvessel cells, both normal ovarian epithelia and ovarian tumor tissuewere examined by immunohistochemistry using the polyclonal antiserum(described above). All 14 ovarian tumors showed positive staining ofSCCE, whereas normal ovarian surface epithelium showed very weakexpression of SCCE antigen (FIG. 10A). 8 of 10 serous adenocarcinomas, 1of 1 mucinous adenocarcinoma, and 2 of 2 clear cell carcinomas showed 2+positive staining (more than 50% of positive tumor cells) of SCCE (Table7). FIGS. 10C and 10E show that stratum corneum chymotrytic enzymestaining is localized to the cytoplasm and the cell membrane of ovariantumor cells. The negative control of each case was also performed,wherein the result showed no nonspecific staining of stratum corneumchymotrytic enzyme (FIGS. 10B, 10D and 10F) and staining of normalovarian epithelial cells which showed little SCCE expression (FIG. 10A).

TABLE 7 Immunohistochemical Expression of SCCE Protein in Normal Ovaryand Ovarian Tumor Lab No. Histology SCCE normal ovary weak + normalovary weak + normal ovary weak + normal ovary weak + normal ovary weak +normal ovary weak + 1036 mucinous LMP + 475 serous carcinoma + 465serous carcinoma ++ 464 serous carcinoma ++ 1039 serous carcinoma ++ 960serous carcinoma ++ 962 serous carcinoma ++ 1551 serous carcinoma ++1813 serous carcinoma ++ 1817 serous carcinoma + 1819 serous carcinoma++ 1244 mucinous carcinoma ++ 947 clear cell carcinoma ++ 948 clear cellcarcinoma ++

EXAMPLE 16

Summary of Known Proteases Detected herein

Most of the above-listed proteases were identified from thesense-His/antisense-Ser primer pair, yielding a 500 bp PCR product (FIG.1, Lane 4). Some of the enzymes are familiar, a short summary of eachfollows.

Hepsin

Hepsin is a trypsin-like serine protease cloned from hepatoma cells.Hepsin is an extracellular protease (the enzyme includes a secretionsignal sequence) which is anchored in the plasma membrane by its aminoterminal domain, thereby exposing its catalytic domain to theextracellular matrix. Hepsin has also been shown to be expressed inbreast cancer cell lines and peripheral nerve cells. Hepsin has neverbefore been associated with ovarian carcinoma. Specific primers for thehepsin gene were synthesized and the expression of Hepsin examined usingNorthern blots of fetal tissue and ovarian tissue (both normal andovarian carcinoma).

FIG. 11A shows that hepsin was expressed in ovarian carcinomas ofdifferent histologic types, but not in normal ovary. FIG. 11B shows thathepsin was expressed in fetal liver and fetal kidney as anticipated, butat very low levels or not at all in fetal brain and lung. FIG. 11C showsthat hepsin overexpression is not observed in normal adult tissue.Slight expression above the background level is observed in the adultprostate. The mRNA identified in both Northern blots was the appropriatesize for the hepsin transcript. The expression of hepsin was examined in10 normal ovaries and 44 ovarian tumors using specific primers toβ-tubulin and hepsin in a quantitative PCR assay, and found it to belinear over 35 cycles. Expression is presented as the ratio of³²P-hepsin band to the internal control, the ³²P-β-tubulin band.

Hepsin expression was investigated in normal (N), mucinous (M) andserous (S) low malignant potential (LMP) tumors and carcinomas (CA).FIG. 12A shows quantitative PCR of hepsin and internal controlβ-tubulin. FIG. 12B shows a bar graph of expression of hepsin in 10normal ovaries and 44 ovarian carcinoma samples.

Hepsin mRNA is highly overexpressed in most histopathologic types ofovarian carcinomas including some low malignant potential tumors (seeFIGS. 12A & 12B). Most noticeably, hepsin is highly expressed in serous,endometrioid and clear cell tumors tested. It is highly expressed insome mucinous tumors, but it is not overexpressed in the majority ofsuch tumors.

Stratum Corneum Chymotrypsin Enzyme (SCCE)

The PCR product identified was the catalytic domain of thesense-His/antisense-Ser of the SCCE enzyme. This extracellular proteasewas cloned, sequenced and shown to be expressed on the surface ofkeratinocytes in the epidermis. SCCE is a chymotrypsin-like serineprotease whose function is suggested to be in the catalytic degradationof intercellular cohesive structures in the stratum corneum layer of theskin. This degradation allows continuous shedding (desquamation) ofcells from the skin surface. The subcellular localization of SCCE is inthe upper granular layer in the stratum corneum of normalnon-palmoplantar skin and in the cohesive parts of hypertrophic plantarstratum corneum. SCCE is exclusively associated with the stratum corneumand has not so far been shown to be expressed in any carcinomatoustissues.

Northern blots were probed with the PCR product to determine expressionof SCCE in fetal tissue and ovarian carcinoma (FIGS. 7A, 7B and 7C).Noticeably, detection of SCCE messenger RNA on the fetal Northern wasalmost non-existent (a problem with the probe or the blot was excludedby performing the proper controls). A faint band appeared in fetalkidney. On the other hand, SCCE mRNA is abundant in the ovariancarcinoma mRNA (FIG. 7A). Two transcripts of the correct size areobserved for SCCE. The same panel of cDNA used for hepsin analysis wasused for SCCE expression.

No SCCE expression was detected in the normal ovary lane of the Northernblot. A comparison of all candidate genes, including a loading marker(β-tubulin), was shown to confirm that this observation was not a resultof a loading bias. Quantitative PCR using SCCE primers, along withβ-tubulin internal control primers, confirmed the overexpression of SCCEmRNA in carcinoma of the ovary with no expression in normal ovariantissue (FIG. 6). FIG. 8 shows the ratio of SCCE to the β-tubulininternal standard in 10 normal and 44 ovarian carcinoma tissues. Again,it is observed that SCCE is highly overexpressed in ovarian carcinomacells. It is also noted that some mucinous tumors overexpress SCCE, butthe majority do not.

Protease M

Protease M was identified from subclones of the His--ser primer pair.This protease was cloned by Anisowicz, et al., and shown to beoverexpressed in carcinomas. A evaluation indicates that this enzyme isoverexpressed in ovarian carcinoma (FIG. 13).

Cofactor I and Complement Factor B

Several serine proteases associated with the coagulation pathway werealso subcloned. Examination of normal and ovarian carcinomas byquantitative PCR for expression of these enzymes, it was noticeable thatthis mRNA was not clearly overexpressed in ovarian carcinomas whencompared to normal ovarian tissue. It should be noted that the samepanel of tumors was used for the evaluation of each candidate protease.

EXAMPLE 17

Summary of Previously Unknown Proteases Detected herein TADG-12

TADG-12 was identified from the primer pairs, sense-His/antisense-Asp(see FIG. 1, Lanes 1 & 2). Upon subcloning both PCR products in lane 2,the 200 bp product had a unique protease-like sequence not included inGenBank. This 200 bp product contains many of the conserved amino acidscommon for the His-Asp domain of the family of serine proteins. Thesecond and larger PCR product (300 bp) was shown to have a high degreeof homology with TADG-12 (His-Asp sequence), but also containedapproximately 100 bp of unique sequence. Synthesis of specific primersand the sequencing of the subsequent PCR products from three differenttumors demonstrated that the larger PCR product (present in about 50% ofovarian carcinomas) includes an insert of about 100 bp near the 5′ end(and near the histidine) of the sequence. This insert may be a retainedgenomic intron because of the appropriate position of splice sites andthe fact that the insert does not contain an open reading frame (seeFIG. 14). This suggests the possibility of a splice site mutation whichgives rise to retention of the intron, or a translocation of a sequenceinto the TADG-12 gene in as many as half of all ovarian carcinomas.

TADG-13 and TADG-14

Specific primers were synthesized for TADG-13 and TADG-14 to evaluateexpression of genes in normal and ovarian carcinoma tissue. Northernblot analysis of ovarian tissues indicates the transcript for theTADG-14 gene is approximately 1.4 kb and is expressed in ovariancarcinoma tissues (FIG. 15A) with no noticeable transcript presence innormal tissue. In quantitative PCR studies using specific primers,increased expression of TADG-14 in ovarian carcinoma tissues was notedcompared to a normal ovary (FIG. 15B). The presence of a specific PCRproduct for TADG-14 in both an HeLa library and an ovarian carcinomalibrary was also confirmed. Several candidate sequences corresponding toTADG-14 have been screened and isolated from the HeLa library.

Clearly from sequence homology, these genes fit into the family ofserine proteases. TADG-13 and TADG-14 are, however, heretoforeundocumented genes which the specific primers of the invention allow tobe evaluated in normal and tumor cells, and with which the presence orabsence of expression of these genes is useful in the diagnosis ortreatment selection for specific tumor types.

PUMP-1

In a similar strategy using redundant primers to metal binding domainsand conserved histidine domains, a differentially expressed PCR productidentical to matrix metallo-protease 7 (MMP-7) was identified, hereincalled PUMP-1. Using specific primers for PUMP-1, PCR produced a 250 bpproduct for Northern blot analysis.

MMP-7 or PUMP-1 is differentially expressed in fetal lung and kidneytissues. FIG. 16A compares PUMP-1 expression in normal ovary andcarcinoma subtypes using Northern blot analysis. Notably, PUMP-1 isexpressed in ovarian carcinoma tissues, and again, the presence of atranscript in normal tissue was not detected. FIG. 16B shows theexpression of PUMP-1 in human fetal tissue, while no transcript could bedetected in either fetal brain or fetal liver. FIG. 16C shows thatPUMP-1 overexpression is not observed in normal adult tissue.Quantitative PCR comparing normal versus ovarian carcinoma expression ofthe PUMP-1 mRNA indicates that this gene is highly expressed in serouscarcinomas, including most low malignant serous tumors, and is, again,expressed to a lesser extent in mucinous tumors (FIGS. 17A & 17B).PUMP-1, however, is so far the protease most frequently foundoverexpressed in mucinous tumors (See Table 8 below).

Cathepsin-L

Using redundant cysteine protease primers to conserved domainssurrounding individual cysteine and histidine residues, the cathepsin-Lprotease was identified in several serous carcinomas. An initialexamination of the expression of cathepsin L in normal and ovarian tumortissue indicates that transcripts for the cathepsin-L protease arepresent in both normal and tumor tissues (FIG. 18). However, itspresence or absence in combination with other proteases of the presentinvention permits identification of specific tumor types and treatmentchoices.

CONCLUSION

Redundant primers to conserved domains of serine, metallo-, and cysteineproteases have yielded a set of genes whose mRNAs are overexpressed inovarian carcinoma. The genes which are clearly overexpressed include theserine proteases hepsin, SCCE protease M, TADG12, TADG14 and themetallo-protease PUMP-1 (see FIG. 19 and Table 8). Northern blotanalysis of normal and ovarian carcinoma tissues indicatedoverexpression of hep sin, SCCE, PUMP-1 and TADG-14. A β-tubulin probeto control for loading levels was included.

TABLE 8 Overexpression of Proteases in Ovarian Tumors Type N Hepsin SCCEPump-1 Protease M Normal 10  0% (0/10) 0% (0/10) 0% (0/10) 0% (0/10) LMP12  58.3% (7/12) 66.7% (8/12) 75.0% (9/12) 75% (9/12) serous 7 85.7%(6/7) 85.7% (6/7) 85.7% (6/7) 100% (7/7) mucinous 5 20.0% (1/5) 40.0%(2/5) 60% (3/5) 40.0% (2/5) Carcinoma 32  84.4% (27/32) 78.1% (25/32)81.3% (26/32) 90.6% (29/32) serous 19  94.7% (18/19) 89.5% (17/19) 78.9%(15/19) 94.7% (18/19) mucinous 7 42.9% (3/7) 28.6% (2/7) 71.4% (5/7)85.7% (6/7) endometr. 3 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)clear cell 3 100% (3/3) 100% (3/3) 100% (3/3) 67.7% (2/3)

DISCUSSION

For the most part, these proteins previously have not been associatedwith the extracellular matrix of ovarian carcinoma cells. No panel ofproteases which might contribute to the growth, shedding, invasion andcolony development of metastatic carcinoma has been previouslydescribed, including the three new candidate serine proteases which areherein disclosed. The establishment of an extracellular protease panelassociated with either malignant growth or malignant potential offersthe opportunity for the identification of diagnostic or prognosticmarkers and for therapeutic intervention through inhibition or downregulation of these proteases.

The availability of the instant gene-specific primers coding for theappropriate region of tumor specific proteases allows for theamplification of a specific cDNA probe using Northern and Southernanalysis, and their use as markers to detect the presence of the cancerin tissue. The probes also allow more extensive evaluation of theexpression of the gene in normal ovary versus low malignant potentialtumor, as well as both high- and low-stage carcinomas. The evaluation ofa panel of fresh frozen tissue from all the carcinoma subtypes (Table 4)allowed the determination of whether a protease is expressedpredominantly in early stage disease or within specific carcinomasubtypes. It was also determined whether each gene's expression isconfined to a particular stage in tumor progression and/or is associatedwith metastatic lesions. Detection of specific combinations of proteasesis an identifying characteristic of the specific tumor types and yieldsvaluable information for diagnoses and treatment selection. Particulartumor types may be more accurately diagnosed by the characteristicexpression pattern of each specific tumor.

Specifically, the present invention utilizes primers to the conservedcatalytic triad domain of the serine protease family (viz.His--Asp--Ser). Using such a strategy to display serine proteasetranscripts found in abundance in carcinoma tissues, with little or noexpression in normal ovary, SCCE gene was detected.

The overall expectation of the search was to identify cell surface orsecreted products which may promote either tumor growth or metastasis.Confirmation of the presence of SCCE (a secreted serine protease) inovarian tumors was indicated initially by subcloning and sequencing PCRproducts derived from amplification of tumor cDNA using redundant primesto the histidine (sense) and the serine (antisense) conserved domains ofthe serine protease catalytic sequences. Characterization of the SCCEprotease (Egelrud, T. J. Invest Dermatol 101, 200-204, 1993) indicatedthat the cohesion between individual corneocytes in the stratum comeurn,the primary substrate for cellular desquamation or shedding of skincells may be degraded by SCCE. Proteolysis of these intercellularmatrices is one of the major events preceding desquamation. SCCE hasonly been identified in the stratum comeurn (Egelrud, T. J InvestDermatol 101, 200-204, 1993; Hansson, et al., J Biol Chem 269,19420-19426, 1994) and immunohistochemical studies confirmed its uniquetissue specific expression by the epithelial cells of the stratumcomeurn (SondeIl, et al., J Histochem Cytochem 42, 459-465, 1994). Itwas therefore surprising to discover that this highly conservedexpression of SCCE to skin is obviated when transformation andcarcinogenesis of ovarian epithelial cells occurs. The clearlydistinctive pattern of expression in both low malignant potential tumorsand overt carcinomas of the ovary over normal ovarian tissue suggeststhat the SCCE protease may also play a role in shedding or desquamationof ovarian tumor cells. This association is especially well preserved inserous adenocarcinomas where disease progression is characterized byearly foci of peritoneal metastasis and which may be the result of anearly overexpression of enzymes such as SCCE and consequent tumor cellshedding. Because SCCE and other proteases (e.g. hepsin) areoverexpressed in ovarian tumors (again with particularly highoverexpression in serous tumors) it seems likely that a concert of lyticactivity at the cell surface may be involved in malignant potential.Several aspects of the tumorigenic process can be dissected andidentified as component parts of such a surface protease concert viz 1)initial expansion of newly transformed cells into the surrounding matrixof supporting tissue of the primary organ; 2) desquamation or sheddingof tumor cells into the surrounding environment; 3) invasion of basementmembrane of the target organ of metastasis; and 4) activation ofmitogenic and angiogenic factors to support the newly establishedmetastatic colony.

While it is not yet clear which proteases are the primary agents in eachof these malignant progression steps, the data here indicate thepotential for the involvement of SCCE in the shedding or desquamationphase of this progression. Certain other factors remain to be resolvedeven with regard to SCCE involvement in tumor cell shedding whichinclude activation of SCCE by proteolysis or cleaving of theaminoterminal peptide of the pro-protease. Furthermore, anantileukoprotease which specifically inhibits SCCE activity has beenrecently identified (Wiedow, O. (1995) Isolierung und Charakterisierungvon Serinprotease Inhibitoren der menschlichen Epidermis, Köster,Berlin). The presence of such an inhibitor might effectively inhibitshedding or desquamation of tumor cells as it has been shown to inhibitthe detachment of corneocytes of keratinized skin tissue.

While there remains an intricate interaction between surface proteaseexpression/activation and/or inhibition, it appears likely that aconcert of enzymes which contribute to tumor growth and spread provide amechanism for such a progression. SCCE expression on ovarian tumor cellsurfaces can provide one mechanism by which tumor cells may be shedearly in the tumor progression process of serous carcinomas.

The unique presence of this protease to keratinized stratum comeum andthe present data showing lack of transcript presence in all normal adultand fetal tissues examined support the potential of this secretedextracellular enzyme as a useful marker for ovarian carcinoma. The factthat inhibition of such an activity prevents normal desquamation of skincells also points to the potential of SCCE as a target for inhibition ordown regulation in therapeutic intervention in the spread or metastasisof ovarian carcinoma.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

1. A method of producing activated T cells directed toward stratumcorneum chymotrytic enzyme (SCCE) comprising the steps of: exposingdendritic cells to a humam SCCE peptide selected from the groupconsisting of SEQ ID Nos. 32, 33, 35, 36, 86 or the human SCCE proteinencoded by the DNA of SEQ ID NO: 30, thereby producing activateddendritic cells; and exposing said activated dendritic cells to T cells,wherein said activated dendritic cells would present said SCCE peptideto said T cells, thereby producing activated T cells directed towardsaid SCCE.